On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.     

Identification of GntR as regulator of the glucose metabolism in Pseudomonas aeruginosa

In contrast to Escherichia coli, glucose metabolism in pseudomonads occurs exclusively through the Entner‐Doudoroff (ED) pathway. This pathway, as well as the three routes to generate the initial ED pathway substrate, 6‐phosphogluconate, is regulated by the PtxS, HexR and GtrS/GltR systems. With GntR (PA2320) we report here the identification of an additional regulator in Pseudomonas aeruginosa PAO1. GntR repressed its own expression as well as that of the GntP gluconate permease. In contrast to PtxS and GtrS/GltR, GntR did not modulate expression of the toxA gene encoding the exotoxin A virulence factor. GntR was found to bind to promoters P_gntR and P_gntP and the consensus sequence of its operator was defined as 5′‐AC‐N‐AAG‐N‐TAGCGCT‐3′. Both operator sites overlapped with the RNA polymerase binding site and we show that GntR employs an effector mediated de‐repression mechanism. The release of promoter bound GntR is induced by gluconate and 6‐phosphogluconate that bind with similar apparent affinities to the GntR/DNA complex. GntR and PtxS are paralogous and may have evolved from a common ancestor. The concerted action of four regulatory systems in the regulation of glucose metabolism in Pseudomonas can be considered as a model to understand complex regulatory circuits in bacteria.     

Humanization of Chicken-Derived scFv Using Yeast Surface Display and NGS Data Mining

Generation of high-affinity monoclonal antibodies by immunization of chickens is a valuable strategy, particularly for obtaining antibodies directed against epitopes that are conserved in mammals. A generic procedure is established for the humanization of chicken-derived antibodies. To this end, high-affinity binders of the epidermal growth factor receptor extracellular domain are isolated from immunized chickens using yeast surface display. Complementarity determining regions (CDRs) of two high-affinity binders are grafted onto a human acceptor framework. Simultaneously, Vernier zone residues, responsible for spatial CDR arrangement, are partially randomized. A yeast surface display library comprising ≈300 000 variants is screened for high-affinity binders in the scFv and Fab formats. Next-generation sequencing discloses humanized antibody variants with restored affinity and improved protein characteristics compared to the parental chicken antibodies. Furthermore, the sequencing data give new insights into the importance of antibody format, used during the humanization process. Starting from the antibody repertoire of immunized chickens, this work features an effective and fast high-throughput approach for the generation of multiple humanized antibodies with potential therapeutic relevance.     

The HBM domain: Introducing bimodularity to bacterial sensing

We have recently reported the three dimensional structure of the McpS chemoreceptor sensor domain in complex with its cognate ligands. The domain was characterized by a bimodular architecture, where ligand binding to each module caused a chemotactic response. This is a novel small molecule binding domain, which, however, is un‐annotated in relevant databases. We report here the domain signature of the family of McpS‐like sensor domains, which was termed helical bimodular (HBM) domain. The HBM domain was identified in Bacteria and Archaea and forms part of chemoreceptors and histidine kinases. The conservation of amino acids in the ligand binding sites of both modules suggests that HBM family members recognize similar ligands.     

Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M_r value 56 000 (80–90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10–20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit M_r value of 52 000 which was close to the M_r value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1–2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD⁺ and was more sensitive to product inhibition by NADH. It exhibited no activity with NADP⁺ as cofactor nor was it inhibited by the presence of high concentrations of NADP⁺ or NADPH. Antibodies to the mitochondrial enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - PDC pyruvate dehydrogenase complex - OGDC 2-oxoglutarate dehydrogenase complex - GDC glycine decarboxylase complex - SDS-PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - TDP thiamine diphosphate - M_r relative molecular mass J.G.L. is grateful to the Biotechnology and Biological Sciences Research Council (BBSRC), U.K. for continuing financial support. M.C. is the holder of a BBSRC-funded earmarked Ph.D. studentship.     

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae , multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.     

Specific gamma‐aminobutyrate chemotaxis in pseudomonads with different lifestyle

The PctC chemoreceptor of Pseudomonas aeruginosa mediates chemotaxis with high specificity to gamma‐aminobutyric acid (GABA). This compound is present everywhere in nature and has multiple functions, including being a human neurotransmitter or plant signaling compound. Because P. aeruginosa is ubiquitously distributed in nature and able to infect and colonize different hosts, the physiological relevance of GABA taxis is unclear, but it has been suggested that bacterial attraction to neurotransmitters may enhance virulence. We report the identification of McpG as a specific GABA chemoreceptor in non‐pathogenic Pseudomonas putida KT2440. As with PctC, GABA was found to bind McpG tightly. The analysis of chimeras comprising the PctC and McpG ligand‐binding domains fused to the Tar signaling domain showed very high GABA sensitivities. We also show that PctC inactivation does not alter virulence in Caenorhabditis elegans. Significant amounts of GABA were detected in tomato root exudates, and deletion of mcpG reduced root colonization that requires chemotaxis through agar. The C. elegans data and the detection of a GABA receptor in non‐pathogenic species indicate that GABA taxis may not be related to virulence in animal systems but may be of importance in the context of colonization and infection of plant roots by soil‐dwelling pseudomonads.     

The carbon count of 2000 years of rice cultivation

More than 50% of the world's population feeds on rice. Soils used for rice production are mostly managed under submerged conditions (paddy soils). This management, which favors carbon sequestration, potentially decouples surface from subsurface carbon cycling. The objective of this study was to elucidate the long‐term rates of carbon accrual in surface and subsurface soil horizons relative to those of soils under nonpaddy management. We assessed changes in total soil organic as well as of inorganic carbon stocks along a 2000‐year chronosequence of soils under paddy and adjacent nonpaddy management in the Yangtze delta, China. The initial organic carbon accumulation phase lasts much longer and is more intensive than previously assumed, e.g., by the Intergovernmental Panel on Climate Change (IPCC). Paddy topsoils accumulated 170–178 kg organic carbon ha^?1 a^?1 in the first 300 years; subsoils lost 29–84 kg organic carbon ha^?1 a^?1 during this period of time. Subsoil carbon losses were largest during the first 50 years after land embankment and again large beyond 700 years of cultivation, due to inorganic carbonate weathering and the lack of organic carbon replenishment. Carbon losses in subsoils may therefore offset soil carbon gains or losses in the surface soils. We strongly recommend including subsoils into global carbon accounting schemes, particularly for paddy fields.